The Kv2.1 K+ channel is the most abundantly expressed and widely distributed voltage-gated K+ channel in mammals. Our previous research demonstrates that in addition to functioning as a delayed rectifier K+ channel and regulating plasma membrane potential, a non-conducting, majority population of Kv2.1 forms endoplasmic reticulum/plasma membrane (ER/PM) contact sites. In hippocampal neurons Kv2.1 channel binding to the cortical endoplasmic reticulum generates micron-sized Kv2.1 clusters on the surface of the soma, proximal dendrites and axon initial segment. Data in the literature indicate that ER/PM junctions regulate neuronal burst firing, the non-vesicular lipid transfer directly from the ER to the cell surface, and plasma membrane PIP2 levels. Our preliminary data show that the Kv2.1-induced ER/PM junctions, but not other ER/PM junctions, alter ER Ca2+ homeostasis, plasma membrane organization, and exocytosis. Interestingly, Kv2.1 interaction with the cortical ER is regulated by neuronal activity and stroke-like insults such as hypoxia, ischemia and excess glutamate, indicating that the functions linked to these microdomains are remodeled following hyperactivity or neuronal insult. Thus, the proposed research examines a novel non-conducting function of Kv2.1 that 1) is central to neuronal physiology and 2) is regulated by neuronal activity, insult and stroke. The three Specific Aims will address the molecular mechanisms by which Kv2.1 alters ER Ca2+ homeostasis and membrane protein localization at somatic ER/PM junctions and exocytosis at presynaptic ER/PM contacts. Aim 1. To test the hypothesis that Kv2.1-induced ER/PM contact sites enhance store-operated Ca2+ entry by providing localized K+ conductance. Preliminary data suggest that ER Ca2+ refilling is enhanced in neurons expressing Kv2.1. Aim 2. To test the hypothesis that the concerted action of Kv2.1 and cortical actin controls the localization of Ca2+ signaling proteins in the vicinity of ER/PM junctions. Preliminary data indicate Kv2.1-induced ER/PM junctions influence the cell surface distribution of Cav1.2, BK K+ channels and b2 adrenergic receptors. Aim 3. To test the hypothesis that synaptic vesicle exocytosis is modulated by Kv2.1 channels at the ER/PM junction in presynaptic terminals. Preliminary data demonstrate that both endogenous and transfected Kv2.1 is localized at presynaptic terminals and that shRNA-based knockdown of Kv2.1 suppresses glutamatergic vesicle exocytosis by 50% without affecting the action potential. While Kv2.1 point mutations that cause human epileptic encephalopathy alter channel conductance, a subset of point mutants that are linked to developmental delay induce premature stop codons in the channel C-terminus that should not affect conductance. Instead, these mutations are predicted to only prevent Kv2.1 binding to the cortical ER. Thus, mutations affecting both the conductance and cortical ER remodeling roles of Kv2.1 underlie human disease. The research in this proposal will substantially advance our understanding of the role that Kv2.1- containing ER/PM contact sites play in neuronal physiology.